The role of iglC gene in intracellular life of Francisella Tularensis

CILJEVI Odrediti ulogu iglC gena u unutarstanicnom životu F. tularensis subsp. novicida u humanim makrofagima i amebama putem kinetike rasta, modulacije apoptoze, biogeneze fagosoma i ultrastrukturalne organizacije stanica nakon infekcije. METODE Stanice A. castellanii i H. vermiformis te humani makrofagi inficirane su s F. tularensis subsp. novicida i njenom mutantom iglC. U odreenim vremenskim periodima nakon infekcije stanice su procesuirane za odreivanje ukupnog broja bakterija metodom kinetike rasta, stupnja zakiseljavanja fagosoma metodom konfokalne mikroskopije dok je ultrastrukturalana organizacija stanice odreivana elektronskom mikroskopijom. Otpornost na apoptozu te aktivacija kaspaze-1 i 3 u humanih makrofaga inficiranih s F. tularensis subsp. novicida ili iglC mutantom odreivana je imunofluorescentim bojenjem putem konfokalane mikroskopije. REZULTATI F. tularensis subsp. novicida pokrece jezgrinu translokaciju p65 podjedinice NF-B u humanim makrofagima, no translokacija NF-B je defektna kod iglC mutante. Iako se kaspaza-1 i kaspaza-3 pokrecu tijekom ranih stadija infekcije humanih makrofaga s F. tularensis subsp. novicida, stanicna smrt je odgoena, što je u korelaciji sa simultanom aktivacijom NF-B. F. tularensis subsp. novicida razmnožava se unutar ameba u fagosomu u kojem nije došlo do acidifikacije dok je iglC mutanta defektna za razmnožavanje u ovim dvjema amebama. ZAKLJUCCI Ovo je istraživanje prvi puta pokazalo simultanu modulaciju kaspaze-1, kaspaze-3 i antiapoptoticnog jezgrinog transkripcijskog cimbenika NF-B i osjetljivu ravnotežu meu njima kako bi se ocuvala sposobnost za život inficirane stanice. Francisella može rasti i preživjeti unutar amebe, a iglC je potreban za unutarstanicni rast u A. castellanii i H. vermiformis. Za razliku od stanica sisavaca gdje se bakterija razmnožava u citoplazmi u amebama se F. tularensis subsp. novicida razmnožava u fagosomu.AIMS To determine the role of the iglC gene in intracellular life of F. tularensis subsp. novicida within human macrophages and amoeba through growth kinetics, modulation of apoptosis, biogenesis of the phagosome and ultrastructural organisation of the cells after the infection. METHODS The cells of A. castellanii, H. vermiformis and human macrophages were infected with F. tularensis subsp. novicida and its mutant iglC. In certain time periods after infection the cells were processed in order to determine colony forming units by growth kinetics, to examine the level of phagosome acidification by confocal microscopy and ultrastructural organisation of the cell was determined by electron microscopy. VII Resistance to apoptosis and activation of caspase-1 and caspase-3 in human macrophages infected with F. tularensis subsp. novicida or iglC mutant was determined with immunoflourescence staining by confocal microscopy. RESULTS F. tularensis triggers nuclear translocation of the p65 subunit of NF-B in hMDMs, but sustained nuclear translocation of NF-B is defective in the iglC mutant. Although caspase-1 and caspase-3 are triggered within F. tularensis-infected hMDMs during early stages of infection, cell death is delayed, which is correlated with simultaneous activation of NF-B. In vitro studies showed intracellular replication of F. tularensis subsp. novicida within A. castellanii and H. vermiformis, but iglC mutant is defective for survival and replication in these amoeba. CONCLUSIONS This study, for the first time showed simultaneous modulation of caspase-1, caspase-3 and the anti-apoptosis nuclear transcription factor NF-B and sensitive balance among them in order to preserve capability for survival of the infected cells. Francisella is capable to grow and survive in amoeba, and iglC is necessary for intracellular growth in A. castellanii and H. vermiformis. In contrast to mammalian cells where bacteria replicate in the cytosol in amoeba F. tularensis subsp. novicida replicates within pahgosomes